Optimized method for antibody capturing by mixed mode chromatography

ABSTRACT

Herein is reported a method for the purification of an antibody directly captured from clarified cell culture supernatants using Streamline CST and/or Capto MMC, wherein especially product related (aggregates and fragments) and process related impurities (host cell protein, media components) could efficiently be removed, resulting in a preparation with a purity comparable to classical protein A affinity chromatography.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation of application Ser. No. 13/883,243, filed May 2,2013, which is a 35 U.S.C. § 371 national stage application ofInternational Patent Application PCT/EP2011/069202 (publishedWO2012/059495), filed Nov. 2, 2011, which claims priority of EuropeanApplication 10190192.4, filed Nov. 5, 2010. The entire disclosure ofeach prior mentioned application is hereby incorporated by reference.

Herein is reported a method for the purification of an antibody directlycaptured from clarified cell culture supernatants using Streamline CSTand/or Capto MMC, wherein especially product related (aggregates andfragments) and process related impurities (host cell protein, DNA, mediacomponents) could efficiently be removed, resulting in a preparationwith a purity comparable to classical protein A affinity chromatography.

BACKGROUND OF THE INVENTION

Protein A chromatography is routinely employed as a first capture stepin industrial monoclonal antibody purification processes due to its highselectivity, resulting in good overall yields and purities. However, amajor drawback of this affinity chromatography is its high price, whichespecially in case of therapeutic antibodies needed at high doses and/orfor chronic administration can account for a significant cost of goodsfactor. In addition, leached protein A ligand from the affinity matrixmust be removed by further chromatography steps due to its potentialimmunogenicity.

Mixed mode chromatography on multimodal resins exhibiting ionic andhydrophobic functionalities can offer a valuable alternative to theclassical affinity approach. Due to the salt tolerability of thehydrophobic component, in most cases a direct loading of the clarifiedcell culture supernatant on the matrix is possible resulting in aneffective capturing the monoclonal antibody. However, due to themultimodal nature of the resin, different types of interaction of theligand with a particular monoclonal antibody are possible, requiringunique wash and elution condition differing from traditionalion-exchange or hydrophobic interaction chromatography.

In WO 2010/080062 a separation method using single polymer phase systemsis reported. A manufacturing process for the production of polypeptidesexpressed in insect cell lines is reported in WO 2008/073620.

SUMMARY OF THE INVENTION

It has been found that a multimodal weak cation exchange chromatographymaterial can be used as first step in a column chromatographypurification method directly with crude cell culture cultivationsupernatant.

One aspect reported herein is a method for producing an anti-IGF-1Rantibody comprising the following steps:

-   -   a) applying a crude mammalian cell culture cultivation        supernatant to a multimodal weak cation exchange chromatography        material,    -   b) recovering the anti-IGF-1R antibody by applying a buffered        solution comprising ethylene glycol and an inorganic salt to the        multimodal weak cation exchange chromatography material and        thereby producing an anti-IGF-1R antibody.

In one embodiment the method comprises the following additional stepa-1) prior to step a):

-   -   a-1) applying a buffered solution comprising an inorganic salt        to the multimodal weak cation exchange chromatography material.

In one embodiment the method comprises the following additional stepa-b) after step a) and prior to step b):

-   -   a-b) applying a buffered solution to the multimodal weak cation        exchange chromatography material, whereby the anti-IGF-1R        antibody is not recovered from the multimodal weak cation        exchange chromatography material.

In one embodiment the step a-b) comprises two steps a-b1) and a-b2):

-   -   a-b1) applying a buffered solution comprising an inorganic salt        to the multimodal weak cation exchange chromatography material,        and    -   a-b2) applying a buffered solution comprising a denaturant to        the multimodal weak cation exchange chromatography material,    -   whereby the anti-IGF-1R antibody is not recovered from the        multimodal weak cation exchange chromatography material.

In one embodiment the denaturant is selected from guanidiniumhydrochloride, and urea.

In one embodiment the inorganic salt is selected from sodium chloride,potassium chloride, and ammonium chloride.

In one embodiment the buffered solution in step b) comprises 20 mM to 30mM Tris, 1050 mM to 1350 mM sodium chloride, and about 20% (w/v)ethylene glycol at a pH value of from pH 7.1 to pH 7.3.

In one embodiment the buffered solution in step a-1) comprises 20 mM to30 mM Tris, and 80 mM to 120 mM sodium chloride at a pH value of from pH7.1 to pH 7.3. In one embodiment the buffered solution in step a-b1)comprises 20 mM to 30 mM Tris, and 80 mM to 120 mM sodium chloride at apH value of from pH 7.1 to pH 7.3. In one embodiment the bufferedsolution in step a-b2) comprises 110 mM to 140 mM Tris, 80 mM to 120 mMsodium chloride, and 30 mM to 40 mM of arginine at a pH value of from pH7.1 to pH 7.3.

In one embodiment the multimodal weak cation exchange chromatographymaterial comprises cross-linked agarose to which a carboxylic acid,ether, thioether and aromatic functional group containing multimodalweak cation exchange ligand is covalently attached.

DETAILED DESCRIPTION OF THE INVENTION

Herein is reported a process for the purification of a monoclonalantibody captured from clarified cell culture supernatants usingStreamline CST and/or Capto MMC comprising optimized conditions forelution and washing. Especially product related (aggregates andfragments) and process related impurities (host cell protein, mediacomponents) can efficiently be removed, resulting in a preparation witha purity comparable to classical affinity chromatography. Thus, with themethod as reported herein a classical protein A affinity chromatographycan be replaced.

In one embodiment the method does not comprise a protein A affinitychromatography step.

General chromatographic methods and their use are known to a personskilled in the art. See for example, Chromatography, 5^(th) edition,Part A: Fundamentals and Techniques, Heftmann, E. (ed.), ElsevierScience Publishing Company, New York, (1992); Advanced Chromatographicand Electromigration Methods in Biosciences, Deyl, Z. (ed.), ElsevierScience BV, Amsterdam, The Netherlands, (1998); Chromatography Today,Poole, C. F., and Poole, S. K., Elsevier Science Publishing Company, NewYork, (1991); Scopes, Protein Purification: Principles and Practice(1982); Sambrook, S., et al. (ed.), Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; or Current Protocols in Molecular Biology, Ausubel,F. M., et al. (eds.), John Wiley & Sons, Inc., New York (1987).

The term “applying to” denotes a partial step of a purification methodin which a solution is brought in contact with a chromatographymaterial. This denotes that either a) the solution is added to achromatographic device in which the chromatography material iscontained, or b) that the chromatography material is added to thesolution. In case a) the solution passes through the device allowing foran interaction between the chromatography material and the substancescontained in solution. Depending on the conditions, such as e.g. pH,conductivity, salt concentration, temperature, and/or flow rate, somesubstances of the solution can be bound to the chromatography materialand other substances can be recovered from the chromatography material.The substances remaining in solution or recovered from thechromatography material can be found in the flow-through. The“flow-through” denotes the solution obtained after the passage of thedevice, which may either be the applied solution or a buffered solution,which is used to wash the column or to cause elution of substances boundto the chromatography material. In one embodiment the device is a columnor a cassette. In case b) the chromatography material can be added, e.g.as a solid, to the solution, e.g. containing the substance of interestto be purified, allowing for an interaction between the chromatographymaterial and the substances in solution. After the interaction thechromatography material is removed, e.g. by filtration, and thesubstance bound to the chromatography material are also removedtherewith from the solution whereas the substances not bound to thechromatography material remain in solution.

The term “bind-and-elute mode” denotes an operation mode of achromatography step, in which a solution containing a substance ofinterest to be purified is applied to a chromatography material, wherebythe substance of interest binds to the chromatography material. Thus,the substance of interest is retained on the chromatography materialwhereas substances not of interest are removed with the flow-through orthe supernatant. The substance of interest is afterwards recovered fromthe chromatography material in a second step with an elution solution.In one embodiment the method as reported herein is operated inbind-and-elute mode.

The term “buffered solution” denotes a solution in which changes of pHdue to the addition or release of acidic or alkaline substances isleveled by the dissolved buffer substance. Any buffer substance withsuch properties can be used. Generally pharmaceutically acceptablebuffers substances are used. In one embodiment the buffered solution isselected from a phosphate buffered solution consisting of phosphoricacid and/or salts thereof, or an acetate buffered solution consisting ofacetic acid and salts thereof, or a citrate buffered solution consistingof citric acid and/or salts thereof, or a morpholine buffered solution,or a 2-(N-morpholino) ethanesulfonic buffered solution, or a histidinebuffered solution, or a glycine buffered solution, or a tris(hydroxymethyl) aminomethane (Tris) buffered solution. In anotherembodiment the buffer solution is selected from a Tris bufferedsolution, or a citrate buffered solution, or a histidine bufferedsolution. The buffered solution may comprise an inorganic salt, such ase.g. sodium chloride, sodium sulphate, potassium chloride, potassiumsulfate, ammonium chloride, or ammonium sulfate.

The terms “continuous elution” and “continuous elution method”, whichare used interchangeably within this application, denote a methodwherein the conductivity of a solution causing elution, i.e. therecovery of a bound compound from a chromatography material, is changed,i.e. raised or lowered, continuously, i.e. the concentration is changedby a sequence of small steps each not bigger than a change of 2%, or of1% of the concentration of the substance causing elution. In this“continuous elution” one or more conditions, for example the pH, theionic strength, concentration of a salt, and/or the flow of achromatography, may be changed linearly or exponentially orasymptotically. In one embodiment the change is linear.

The term “step elution” denotes a method wherein e.g. the concentrationof a substance causing elution, i.e. the recovery of a bound substancefrom a chromatography material, is raised or lowered at once, i.e.directly from one value/level to the next value/level. In this “stepelution” one or more conditions, for example the pH, the ionic strength,concentration of a salt, and/or the flow of a chromatography, can bechanged all at once from a first, e.g. starting, value to a second, e.g.final, value. Thus, the conditions are changed incrementally, i.e.stepwise, in contrast to a linear change.

The term “multimodal weak cation exchange chromatography material”denotes an immobile high molecular weight matrix, such as chemicallycross-linked agarose, that carries covalently bound charged substituentsused as stationary phase in ion exchange chromatography. For overallcharge neutrality not covalently bound counter ions are bound thereto.The “multimodal weak cation exchange chromatography material” has theability to exchange its not covalently bound cationic counter ions forsimilarly charged ions of the surrounding solution. A “multimodal weakcation exchange chromatography material” comprises covalently boundligands that are capable of exerting ionic interactions, hydrophobicinteractions, van-der-Waals interactions as well as the formation ofhydrogen bonds with molecules comprised in a surrounding solution.

To a person skilled in the art procedures and methods are well known toconvert an amino acid sequence, e.g. of a polypeptide, into acorresponding nucleic acid sequence encoding this amino acid sequence.Therefore, a nucleic acid is characterized by its nucleic acid sequenceconsisting of individual nucleotides and likewise by the amino acidsequence of a polypeptide encoded thereby.

The term “under conditions suitable for binding” and grammaticalequivalents thereof as used within this application denotes that asubstance of interest, e.g. an anti-IGF-1R antibody, binds to astationary phase when brought in contact with it, e.g. an ion exchangematerial. This does not necessarily denote that 100% of the substance ofinterest is bound but essentially 100% of the substance of interest isbound, i.e. at least 50% of the substance of interest is bound,preferably at least 75% of the substance of interest is bound,preferably at least 85% of the substance of interest is bound, morepreferably more than 95% of the substance of interest is bound to thestationary phase.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity. Naturally occurring antibodies aremolecules with varying structures. For example, native IgG antibodiesare hetero tetrameric glycoproteins of about 150,000 Daltons, composedof two identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variabledomain (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three or four constant domains (CH1, CH2,CH3 and optionally CH4), Similarly, from N- to C-terminus, each lightchain has a variable domain (VL), also called a variable light domain ora light chain variable domain, followed by a constant light chain (CL)domain. The light chain of an antibody may be assigned to one of twotypes, called kappa (κ) and lambda (λ), based on the amino acid sequenceof its constant domain. Human insulin-like growth factor I receptor(IGF-IR, EC 2.7.112, CD 221 antigen) belongs to the family oftransmembrane protein tyrosine kinases (LeRoith, D., et al., Endocrin.Rev. 16 (1995) 143-163; Adams, T. E., et al., Cell. Mol. Life Sci. 57(2000) 1050-1063). IGF-IR binds IGF-I with high affinity and initiatesthe physiological response to this ligand in vivo. IGF-IR also binds toIGF-II, however with slightly lower affinity. IGF-IR overexpressionpromotes the neoplastic transformation of cells and there existsevidence that IGF-IR is involved in malignant transformation of cellsand is therefore a useful target for the development of therapeuticagents for the treatment of cancer (Adams, T. E., et al., Cell. Mol.Life Sci. 57 (2000) 1050-1063). Exemplary anti IGF-1R antibodies, theirencoding sequences and methods of production are reported in WO2004/087756, WO 2007/045456, and WO 2007/115814.

For the purification of immunoglobulins or immunoglobulin fragments,which have been produced e.g. by cell cultivation methods, generally acombination of different chromatography steps is employed. Normally aprotein A affinity chromatography is followed by one or two additionalseparation steps. In one embodiment said additional chromatography stepsare a cation and an anion exchange chromatography step or vice versa.The final purification step is a so called “polishing step” for theremoval of trace impurities and contaminants like aggregatedimmunoglobulins, residual HCP (host cell protein), DNA (host cellnucleic acid), viruses, or endotoxins. In one embodiment the finalpurification step is an anion exchange chromatography in flow-throughmode.

It has been found that a multimodal weak cation exchange chromatographymaterial can be used as first step in a column chromatographypurification method directly with crude cell culture cultivationsupernatant instead of the commonly used protein A affinitychromatography.

One aspect reported herein is a method for producing an anti-IGF-1Rantibody comprising the following steps:

-   -   a) applying a crude mammalian cell culture cultivation        supernatant to a multimodal weak cation exchange chromatography        material,    -   b) recovering the anti-IGF-1R antibody by applying a buffered        solution comprising ethylene glycol and an inorganic salt to the        multimodal weak cation exchange chromatography material and        thereby producing an anti-IGF-1R antibody.

For the method as reported herein no pre-conditioning of the crudecultivation supernatant is required. This was surprising as forcultivation supernatants obtained from cultivations, in which adifferent antibody has been produced, at least a reduction of theconductivity is required in order to allow a capturing of the antibodydirectly from the culture supernatant. Additionally the capturing of theanti-IGF-1R antibody from the crude cell cultivation supernatant isalmost quantitatively. As the generally applicable conditions for therecovery of polypeptides from cation exchange chromatography materialsand the conditions for binding by hydrophobic interactions to themultimodal weak cation exchange chromatography material point inopposite directed novel conditions for the recovery of an antibody fromthe multimodal weak cation exchange chromatography material were needed.

In one embodiment the method comprises the following additional stepa-1) prior to step a):

-   -   a-1) applying a buffered solution comprising an inorganic salt        to the multimodal weak cation exchange chromatography material.

In another embodiment the method comprises the following additional stepa-b) after step a) and prior to step b):

-   -   a-b) applying a buffered solution to the multimodal weak cation        exchange chromatography material, whereby the anti-IGF-1R        antibody is not recovered from the multimodal weak cation        exchange chromatography material.

In a further embodiment the step a-b) comprises two steps a-b1) anda-b2):

-   -   a-b1) applying a buffered solution comprising an inorganic salt        to the multimodal weak cation exchange chromatography material,        and    -   a-b2) applying a buffered solution comprising a denaturant to        the multimodal weak cation exchange chromatography material,        whereby the anti-IGF-1R antibody is not recovered from the        multimodal weak cation exchange chromatography material.

In also an embodiment the denaturant is selected from guanidiniumhydrochloride, urea, and arginine. In one embodiment the inorganic saltis selected from sodium chloride, potassium chloride, and ammoniumchloride. In also an embodiment the buffered solution in step b)comprises 20 mM to 30 mM Tris, 1050 mM to 1350 mM sodium chloride, andabout 20% (w/v) ethylene glycol at a pH value of from pH 7.1 to pH 7.3.In one embodiment the buffered solution in step a-1) comprises 20 mM to30 mM Tris, and 80 mM to 120 mM sodium chloride at a pH value of from pH7.1 to pH 7.3. In a further embodiment the buffered solution in stepa-b1) comprises 20 mM to 30 mM Tris, and 80 mM to 120 mM sodium chlorideat a pH value of from pH 7.1 to pH 73. In another embodiment thebuffered solution in step a-b2) comprises 110 mM to 140 mM Tris, 80 mMto 120 mM sodium chloride, and 30 mM to 40 mM arginine at a pH value offrom pH 7.1 to pH 7.3. In still another embodiment the multimodal weakcation exchange chromatography material comprises cross-linked agaroseto which a carboxylic acid, ether, thioether and aromatic functionalgroup containing multimodal weak cation exchange ligand is covalentlyattached. In one embodiment a total volume of five column volumes of thebuffered solution in step a-1) is applied to the multimodal weak cationexchange chromatography material. In another embodiment a total volumeof five column volumes of the buffered solution in step a-b1) is appliedto the multimodal weak cation exchange chromatography material. In alsoan embodiment a total volume of ten column volumes of the bufferedsolution in step a-b2) is applied to the multimodal weak cation exchangechromatography material. In still an embodiment a total volume of tencolumn volumes of the buffered solution in step b) is applied to themultimodal weak cation exchange chromatography material.

It has been found that the individual components of the bufferedsolutions have the following effects:

Yield LMWs HMWs Purity Salt NaCl − − 0 + NH4Cl −− − + + ethylene − 0 0 0glycol Denaturant Urea −− − 0 + Arginine − − 0 + +: increase; −:decrease; 0: no effect

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 Analytical size exclusion chromatogram of the anti-IGF-1Rantibody chromatography eluate obtained in step b) of Example 1.

FIG. 2 Elution chromatogram of the anti-IGF-1R antibody obtained with amethod as reported herein according to the conditions denoted in Example3.

FIG. 3 Analytical size exclusion chromatogram of the anti-IGF-1Rantibody chromatography eluate obtained in step b) (a) and for theeluate obtained in step a-b2) (b).

FIG. 4 Elution chromatogram of an application of a crude cultivationsupernatant comprising anti-IL13R antibody.

FIG. 5 Elution chromatogram of an application of a pre conditionedcultivation supernatant comprising anti-IL13R antibody.

EXAMPLES Materials & Methods

If not otherwise indicated have the different methods been performedaccording to the material manufacturer's manual.

Recombinant DNA Techniques:

Standard methods were used to manipulate DNA as described in Sambrook,J., et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Protein Determination:

Protein concentration was determined by determining the optical density(OD) at 280 nm, with a reference wavelength of 320 nm, using the molarextinction coefficient calculated on the basis of the amino acidsequence.

Size-Exclusion-HPLC:

The chromatography was conducted with a Tosoh Haas TSK 3000 SWXL columnon an ASI-100 HPLC system (Dionex, Idstein, Germany). The elution peakswere monitored at 280 nm by a UV diode array detector (Dionex). Afterdissolution of the concentrated samples to 1 mg/ml the column was washedwith a buffer consisting of 200 mM potassium dihydrogen phosphate and250 mM potassium chloride pH 7.0 until a stable baseline was achieved.The analyzing runs were performed under isocratic conditions using aflow rate of 0.5 ml/min. over 30 minutes at room temperature. Thechromatograms were integrated manually with Chromeleon (Dionex, Idstein,Germany).

Reversed Phase HPLC (RP-HPLC):

The purity is analyzed by RP-HPLC. The assay is performed on a Poroshellcolumn using an acetonitrile/aqueous TFA gradient. The elution profileis monitored as UV absorbance at 215 nm. The percentages of the elutedsubstances are calculated based upon the total peak area of the elutedproteins.

DNA-Threshold-System:

see e.g. Merrick, H., and Hawlitschek, G., Biotech Forum Europe 9 (1992)398-403

Host Cell Protein Determination:

The walls of the wells of a micro titer plate are coated with a mixtureof serum albumin and Streptavidin. A goat derived polyclonal antibodyagainst HCP is bound to the walls of the wells of the micro titer plate.After a washing step different wells of the micro titer plate areincubated with a HCP calibration sequence of different concentrationsand sample solution. After the incubation not bound sample material isremoved by washing with buffer solution. For the detection the wells areincubated with an antibody peroxidase conjugate to detect bound hostcell protein. The fixed peroxidase activity is detected by incubationwith ABTS and detection at 405 nm.

Example 1 Chromatography of an Anti-IGF-1R Antibody with a Method asReported Herein on the Multimodal Weak Cation Exchange ChromatographyMaterial Capto™ MMC

The chromatography was performed with the following parameters:

-   -   resin: Capto™ MMC    -   column diameter: 1 cm    -   bed height: 14.6 cm    -   applied solution: crude cell cultivation supernatant    -   load: 30 mg antibody per ml of material    -   step a-1) 25 mM Tris-HCl, 100 mM NaCl, pH 7.1    -   step a-b1) 25 mM Tris-HCl, 100 mM NaCl, pH 7.1    -   step b) 25 mM Tris-HCl, 1200 mM NaCl, 20% (w/v) ethylene glycol,        pH 7.2    -   elution method: step elution    -   flow rate: 250 cm/h

The analytical size exclusion chromatogram of the obtained antibody isshown in FIG. 1.

analytical SEC results of recovered buffered solution of step b)antibody ethylene total total Tris NaCl glycol HMW antibody HMW recovery[mM] [mM] [wt %] pH [%] [%] [%] [%] 25 1200 20 7.2 1.6 91.8 6.7

Example 2 Comparative Chromatographies to Example 1 of an Anti-IGF-1RAntibody with Conditions Different from the Method as Reported Herein onthe Multimodal Weak Cation Exchange Chromatography Material Capto™ MMC

The chromatography was performed with the following parameters:

-   -   resin: Capto™ MMC    -   column diameter: 1 cm    -   bed height: 14.6 cm    -   applied solution: crude cell cultivation supernatant    -   load: 30 mg antibody per ml of material    -   step a-1) 25 mM Tris-HCl, 100 mM NaCl, pH 7.1    -   step a-b1) 25 mM Tris-HCl, 100 mM NaCl, pH 7.1    -   step b) variable—see table below    -   elution method: step elution    -   flow rate: 250 cm/h

From the table presented below it can be seen that with slightlydifferent conditions of step b) the purification could be performed lesseffective.

analytical SEC results of recovered buffered solution of step b)antibody ethylene total total Tris NaCl glycol HMW antibody HMW recovery[mM] [mM] [wt %] pH [%] [%] [%] [%] 300 1500 20 8.9 5.1 85.4 9.5 93 3001500 20 7.1 4.7 85.6 9.7 95 300 1500 0 7.1 3.4 87.0 3.8 90 25 1500 208.9 4.6 86.3 9.2 101 25 1500 20 7.1 3.7 86.8 9.5 95 25 250 20 7.1 2.456.4 41.3 8 25 250 0 7.1 3.5 41.0 45.6 3

Example 3 Chromatography of an Anti-IGF-1R Antibody with a Method asReported Herein on the Multimodal Weak Cation Exchange ChromatographyMaterial Capto™ MMC

The chromatography was performed with the following parameters:

-   -   resin: Capto™ MMC    -   column diameter: 1 cm    -   bed height: 14.6 cm    -   applied solution: crude cell cultivation supernatant    -   load: 30 mg antibody per ml of material    -   step a-1) 25 mM Tris-HCl, 100 mM NaCl, pH 7.1    -   step a-b1) 25 mM Tris-HCl, 100 mM NaCl, pH 7.1    -   step a-b2) 125 mM Tris-HCl, 100 mM NaCl, 38 mM arginine, pH 8.7    -   step b) 25 mM Tris-HCl, 1200 mM NaCl, 20% (w/v) ethylene glycol,        pH 7.2    -   elution method: step elution    -   flow rate: 250 cm/h

The corresponding elution diagram is shown in FIG. 2 and the analyticalsize exclusion chromatogram for step b) in FIG. 3a and for step a-b2) inFIG. 3b ).

analytical SEC results of recovered antibody buffered solution of stepa-b2) total total Tris NaCl arginine LMW antibody HMW recovery [mM] [mM][wt %] pH [%] [%] [%] [%] 125 100 38 8.7 1.6 95.7 2.7 93

Example 4 Comparative Chromatographies to Example 3 of an Anti-IGF-1RAntibody with Conditions Different from the Method as Reported Herein onthe Multimodal Weak Cation Exchange Chromatography Material Capto™ MMC

The chromatography was performed with the following parameters:

-   -   resin: Capto™ MMC    -   column diameter: 1 cm    -   bed height: 14.6 cm    -   applied solution: crude cell cultivation supernatant    -   load: 30 mg antibody per ml of material    -   step a-1) 25 mM Tris-HCl, 100 mM NaCl, pH 7.1    -   step a-b1) 25 mM Tris-HCl, 100 mM NaCl, pH 7.1    -   step a-b2) variable—see table below    -   step b) 25 mM Tris-HCl, 1200 mM NaCl, 20% (w/v) ethylene glycol,        pH 7.2    -   elution method: step elution    -   flow rate: 250 cm/h

From the table presented below it can be seen that with slightlydifferent conditions of step b) the purification could be performed lesseffective.

analytical SEC results of recovered antibody buffered solution of stepa-b2) total total Tris NaCl arginine HMW antibody HMW recovery [mM] [mM][wt %] pH [%] [%] [%] [%] 25 100 40 8.0 4.4 93.7 1.9 100 25 250 40 8.01.4 97.2 1.4 81 25 175 40 8.9 2.2 96.5 1.3 84 162.5 100 40 8.9 2.8 95.41.9 95 162.5 250 40 7.1 1.2 96.9 2.0 57 162.5 175 40 8.0 1.3 97.0 1.7 74162.5 100 40 7.1 2.8 96.1 1.0 80

Example 5 Comparative Example an Anti-IL13R Antibody with NoPre-Conditioning of the Crude Cultivation Supernatant

The chromatography was performed with the following parameters:

-   -   resin: Capto™ MMC    -   column diameter: 1 cm    -   bed height: 10 cm    -   applied solution: crude cell cultivation supernatant    -   load: 21 mg antibody per ml of material    -   step a-1) 70 mM potassium phosphate, pH 7.3, 10.9 mS/cm    -   step a-b) 70 mM potassium phosphate, pH 7.3, 10.9 mS/cm    -   step b) 1 M KCl, pH 3.0, 105.6 mS/cm    -   elution method: step elution    -   flow rate: 150 cm/h

88% of the applied antibody was not bound to the chromatography materialand was found in the flow through (FIG. 4).

Example 6 Comparative Example an Anti-IL13R Antibody withPre-Conditioning of the Crude Cultivation Supernatant

The chromatography was performed with the following parameters:

-   -   resin: Capto™ MMC    -   column diameter: 1 cm    -   bed height: 11 cm    -   applied solution: cell cultivation supernatant adjusted to 1.4        mS/cm    -   load: 20 mg antibody per ml of material    -   step a-1) 10 mM potassium phosphate, pH 6.5, 1.4 mS/cm    -   step a-b) 10 mM potassium phosphate, pH 6.5, 1.4 mS/cm    -   step b) 100 mM potassium phosphate, pH 7.5, 14.9 mS/cm    -   elution method: linear gradient    -   flow rate: 150 cm/h

In the chromatogram (FIG. 5) a sharp peak can be seen. The obtainedantibody had a purity of 96.6% with a yield of 68%.

The invention claimed is:
 1. A method for producing an antibodycomprising the following steps: a) applying a crude mammalian cellculture cultivation supernatant to a multimodal weak cation exchangechromatography material; and b) recovering the antibody by applying abuffered solution comprising ethylene glycol and an inorganic salt tothe multimodal weak cation exchange chromatography material, therebyproducing the antibody.
 2. The method according to claim 1, wherein themethod comprises the following additional step a-1) prior to step a):a-1) applying a buffered solution comprising an inorganic salt to themultimodal weak cation exchange chromatography material.
 3. The methodaccording to claim 1, wherein the method comprises the followingadditional step a-b) after step a) and prior to step b): a-b) applying abuffered solution to the multimodal weak cation exchange chromatographymaterial, whereby the antibody is not recovered from the multimodal weakcation exchange chromatography material.
 4. The method according toclaim 3, wherein the step a-b) comprises two steps a-b1) and a-b2):a-b1) applying a buffered solution comprising an inorganic salt to themultimodal weak cation exchange chromatography material, and a-b2)applying a buffered solution comprising a denaturant to the multimodalweak cation exchange chromatography material, whereby the antibody isnot recovered from the multimodal weak cation exchange chromatographymaterial.
 5. The method according to claim 4, wherein the denaturant isselected from guanidinium hydrochloride, and urea.
 6. The methodaccording to claim 1, wherein the inorganic salt is selected from sodiumchloride, potassium chloride, and ammonium chloride.
 7. The methodaccording to claim 1, wherein the buffered solution in step b) comprises20 mM to 30 mM Tris, 1050 mM to 1350 mM sodium chloride, and about 20%(w/v) ethylene glycol at a pH value of from pH 7.1 to pH 7.3.
 8. Themethod according to claim 2, wherein the buffered solution in step a-1)comprises 20 mM to 30 mM Tris, and 80 mM to 120 mM sodium chloride at apH value of from pH 7.1 to pH 7.3.
 9. The method according to claim 4,wherein the buffered solution in step a-b1) comprises 20 mM to 30 mMTris, and 80 mM to 120 mM sodium chloride at a pH value of from pH 7.1to pH 7.3.
 10. The method according to claim 4, wherein the bufferedsolution in step a-b2) comprises 110 mM to 140 mM Tris, 80 mM to 120 mMsodium chloride, and 30 mM to 40 mM arginine at a pH value of from pH7.1 to pH 7.3.
 11. The method according to claim 1, wherein themultimodal weak cation exchange chromatography material comprisescross-linked agarose to which a carboxylic acid, ether, thioether andaromatic functional group containing multimodal weak cation exchangeligand is covalently attached.